Single-Dose P2 X4R Single-Chain Fragment Variable Antibody Permanently Reverses Chronic Pain in Male Mice

Abstract

Non-opioid single-chain variable fragment (scFv) small antibodies were generated as pain-reducing block of P2X4R receptor (P2X4R). A panel of scFvs targeting an extracellular peptide sequence of P2X4R was generated followed by cell-free ribosome display for recombinant antibody selection. After three rounds of bio-panning, a panel of recombinant antibodies was isolated and characterized by ELISA, cross-reactivity analysis, and immunoblotting/immunostaining. Generated scFv antibodies feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their ~30% smaller size. Two anti-P2X4R scFv clones (95, 12) with high specificity and affinity binding were selected for in vivo testing in male and female mice with trigeminal nerve chronic neuropathic pain (FRICT-ION model) persisting for several months in untreated BALBc mice. A single dose of P2X4R scFv (4 mg/kg, i.p.) successfully, completely, and permanently reversed chronic neuropathic pain-like measures in male mice only, providing retention of baseline behaviors indefinitely. Untreated mice retained hypersensitivity, and developed anxiety- and depression-like behaviors within 5 weeks. In vitro P2X4R scFv 95 treatment significantly increased the rheobase of larger-diameter (>25 µm) trigeminal ganglia (TG) neurons from FRICT-ION mice compared to controls. The data support use of engineered scFv antibodies as non-opioid biotherapeutic interventions for chronic pain.

Neuropathic Pain: Mechanisms, Sex Differences, and Potential Therapies for a Global Problem

“…Male-specific antiallodynia evoked by blocking P2X4Rs pharmacologically, originally observed with TNP-ATP (24), has also been elicited by using a function-blocking anti-P2X4R antibody (30). A single intraperitoneal dose of the anti-P2X4R antibody reversed mechanical allodynia for more than five weeks and prevented the development of anxiety-and depressive-like behaviors.…”

Pathology of pain and its implications for therapeutic interventions

“… 764 P2X4R scFv95 Trigeminal neuralgia Constriction of the trigeminal infraorbital nerve in male and female BALB/c mice scFv95 completely reverses the neuropathic pain only in the males. 765 BDNF R3bH01 Peripheral nerve injury SNI and tibial nerve transection in male SD rats R3bH01 inhibits hyperactivity to heat and mechanical stimuli of sensory neurons in a dose-dependent manner. 766 HMGB1 anti-HMGB1 nAb Trigeminal neuralgia Constriction of distal infraorbital nerve in male ddY mice Anti-HMGB1 nAb mitigates neuropathic pain through blocking activation of immune cells.…”